Method for high frequency tracking and sub-nm sample stabilization in single molecule fluorescence microscopy

Abstract

While fluorescence microscopes and atomic force microscopes are widely used to visualize, track, and manipulate single biomolecules, the resolution of these methods is limited by sample drift. To minimize drift, active feedback methods have recently been used to stabilize single molecule microscopes on the sub-nanometer scale. However, these methods require high intensity lasers which limits their application in single molecule fluorescence measurements. Furthermore, these feedback methods do not track user-defined regions of the sample, but rather monitor the relative displacement of an unknown point on a fiducial marker, which limits their use in biological force measurements. To overcome these limitations, we have developed a novel method to image, track and stabilize a sample using low laser intensities. We demonstrate the capabilities of our approach by tracking a user-chosen point on a fiducial marker at 8.6 kHz and stabilizing it with sub-nanometer resolution. We further showcase the application of our method in single molecule fluorescence microscopy by imaging and stabilizing individual fluorescently-tagged streptavidin proteins under biologically relevant conditions. We anticipate that our method can be easily used to improve the resolution of a wide range of single molecule fluorescence microscopy and integrated force-fluorescence applications.

An alternative framework for fluorescence correlation spectroscopy

“…For the experimental data acquired with elliptical confocal volumes, Cy3 dye and Cy3-labeled streptavidin solutions were prepared by suspending Cy3 or streptavidin in glycerol/buffer (pH 7.5, 10 m m Tris-HCl,100 m m NaCl and 10 m m KCl, 2.5 m m CaCl 2 ) at different v/v, to a final concentration of either 100 p m or 1 n m . The solutions were added onto a glass-bottomed fluid-cell, mounted on a custom designed single-molecule fluorescence confocal microscope 68,69 and a 532 nm laser beam was focused to a diffraction-limited spot on the glass coverslip of the fluid-cell using a × 60, 1.42 NA, oil-immersion objective (Olympus). The laser power was measured before the objective and the beam was reflected by a dichroic and focused by the objective on to the sample.…”

“…A bandpass filter was placed in front of the detector to transmit only the fluorescence from Cy3 and to block the back-scattered excitation light. TTL pulses, triggered by the arrival of individual photons on the SPAD, were timestamped and recorded at 80 MHz by a field programmable gated array (FPGA, NI Instruments) using custom LabVIEW software 69 and initially binned at 100 μs.…”

Pitching Single-Focus Confocal Data Analysis One Photon at a Time with Bayesian Nonparametrics

“…Individual photon arrivals on the detector triggered transistor-transistor-logic pulses and are both time stamped and registered at 80 MHz. This is achieved using a field-programmable gate array (FPGA, NI Instruments) and custom LabVIEW software [74].…”

Robust scan synchronized force-fluorescence imaging

“…However, moving the beam relative to the input of a powerful objective causes optical aberration and the inherent sinusoidal motion of the galvo mirrors causes image distortion. A stationary sample also cannot be stabilized with active feedback, which is useful for monitoring a point of interest over a long period of time and reducing unwanted sample drift (6,7). In contrast, was not certified by peer review) is the author/funder.…”